The killer cell immunoglobulin-like receptors (KIR) are a family of receptors that are expressed on natural killer (NK) cells and some T cells. Known KIR ligands are class I HLA-C and HLA-B (Bw4), which interact with KIR inhibitory or stimulatory receptors to regulate the immunological response, thereby regulating cell execution by NK cells and other cytotoxic cells. A total of 17 KIR loci have been identified, including 15 expressed genes and 2 pseudogenes, with between 2 and 12 alleles seen at each of the polymorphic KIR loci. Diversity in terms of both number and combination of KIR genes exists among individuals, as well as extensive allele polymorphism, all of which affect the strength and breadth of the immune response.
Traditional KIR genotyping methods utilize PCR and sequence-specific priming (SSP) (Dupont et al., Tissue Antigens, 49(6):557-63 (1997); Selvakumar et al., Tissue Antigens, 49(6):564-73 (1997); Uhrberg et al., Immunity, 7(6):753-63 (1997); Shilling et al., Blood, 101(9):3730-3740 (2003); Shilling et al., J Immunol, 168(5):2307-15 (2002)). The SSP assay requires that genomic DNA be amplified using a collection of primers in separate reactions in order to define the various loci or alleles to be detected by fragment lengths using gel electrophoresis. However, there are drawbacks to utilizing the SSP method for higher-throughput analysis of KIR loci in populations. In particular, the KIR SSP amplifications require many different annealing and extension time conditions, which are machine and technologist time intensive, and not conducive to high-throughput analysis. Another significant limitation of the SSP method is that it requires a large quantity of high quality DNA (>5 μg). Furthermore, the SSP method poses the problem of sample amplification failure, which could be due to either general PCR failure or a sequence variant, neither of which can be distinguished utilizing this method. Accordingly, amplification failure could result in erroneous KIR genotyping results.
An alternative KIR genotyping assay uses sequence-specific oligonucleotide probes (SSOP) developed for locus-specific resolution of 14 KIR genes. The SSOP assay requires a small quantity of genomic DNA (50-100 ng) amplified at four KIR domains. PCR products are then denatured and vacuum blotted onto replicate 96-sample nylon membranes. Replicate membranes are hybridized to 39 sequence-specific probes, washed under stringent conditions to remove unbound probe, and developed using non-radioactive detection methods. KIR probe hybridization patterns are then decoded using a computer program. Although generally more efficient than SSP methods, genotyping analysis by SSOP assays is still cumbersome.
Accordingly, there remains a need in the art for a method for performing efficient and reproducible high-throughput genotyping of the KIR locus. The present invention addresses this need.